Isolation and characterization of allergen-binding cells for diagnosis of hypersensitivity

ABSTRACT

Methods and compositions are provided for the diagnosis of allergen hypersensitivity in a patient. Rare, allergen-specific cells are enriched from a complex cell population, e.g. a patient blood sample. The percentage of blood cells that bind to a particular allergen is less than 0.01%. The allergen-specific cell population is enriched by magnetic cell sorting. In normal blood, the allergen-binding cells are primarily B-cells expressing CD19 and CD21. In blood from allergic patients, an additional population of effector cells, e.g. basophilic granulocytes is labeled by the allergen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/660,035, filedJun. 6, 1996, now U.S. Pat. No. 5,786,161.

INTRODUCTION Technical Field

The field of this invention is the diagnosis of allergenhypersensitivity.

Background

Allergy or hypersensitivity of the immune system in its different formsaffects more than 20% of the human population. Man is a highlysusceptible species to anaphylaxis. After sensitization with anallergen, a second exposure elicits constriction of the bronchioles, insome cases resulting in death from asphyxia. This allergic reaction ismediated by allergen-specific antibodies, mostly of the IgE class. Theantibodies can be directed against a variety of antigens, such asmolecules from pollen, fungi, food, house dust mite, hymenoptera venomsor animal danders.

The aggregation of mast cell and basophil high-affinity IgE receptors byIgE and antigen causes the release of mediators and cytokines, includingheparin, eosinophil and neutrophil chemotactic factors, leukotrienes andthromboxanes. Immunoglobulin class switching to IgE expression ismediated by IL-4 or IL-13 and may be mediated through CD40 stimulation.These cytokines may be produced by T helper cells, or by activated mastcell/basophil like cells. Activation of mast cells can provoke anongoing local allergic reaction as long as antigen confrontation ismaintained.

Prophylactic treatment by hyposensitization, or avoidance of theallergen, requires identification of the specific allergen that iscausing the hypersensitive condition. When allergen extracts areadministered intradermally to detect or confirm the allergic status of apatient, an allergic patient will respond with an inflammatory reactionat the site of the injection However, this type of testing isunreliable, and causes significant patient discomfort. Methods ofallergen testing that are less invasive would be highly desirable. Inaddition, the direct analytical and preparative assessment of cells thatspecifically bind with allergens would provide valuable diagnostic toolsand greatly facilitate analysis of the human allergic response.

Relevant Literature

Miltenyi et al. (1990) Cytometry 11:231-236 describe the use of antibodyconjugated superparamagnetic particles for separating rare cellpopulations. Oshiba et al. (1994) Clin. Immunology and Immunopathology72:342-349 describe the isolation of B cells specific for tetanus toxinor KLH hapten by conjugating to magnetic particles. Donhoe et al.(1995), J. Allergy Clin. Immunol. 95:587-596 Analyze IgE⁺ cells inperipheral blood of atopic and hypersensitive donors by two- andthree-color flow cytometry for B cell differentiation markers. Manz etal. (1995) P.N.A.S. 92:1921-1925 describe the analysis and sorting ofcells according to secreted molecules that are trapped on the cellsurface with an affinity matrix. Gross et al. (1995) P.N.A.S. 92:537-541describe the analysis of rare cell populations using flow cytometry.

Surface phenotyping of basophils from peripheral blood on the basis of anegative reactivity with mixed antibodies to CD2, CD14, CD16, and CD19,analyzed by flow cytometry, is described in Takahashi et al. (1993) J.Immunol Methods 162:17-21. Mul et al. (1992) J. Immunol Methods149:207-14 describe the purification of human basophilic granulocyteswith immunomagnetic beads conjugated to monoclonal antibodies specificfor CD2, CD14, CD16 and CD19.

Hoffman (1994) Int. Arch. Allergy Immunol. 104:184-190 provides thecomplete amino acid sequence of two vespid venom phospholipases. Oresteet al. (1991) Int. Arch. Allergy Immunol. 96:19-27 purify andcharacterize the major allergen of Parietaria officinalis. D'Amato etal. (1992) Allergy 47:443-449 review Parietaria pollinosis.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the diagnosis of allergenhypersensitivity in a patient. The diagnosis utilizes a sample ofpatient hematopoietic cells, e.g. blood, and does not require invasiveintradermal challenge. Specific allergen-binding cells are enriched fromthe sample by magnetic cell sorting. The diagnosis relies on differencesin the composition of allergen-binding cells in allergic vs. normalindividuals. In normal blood, the allergen-binding cells are primarilyB-cells expressing CD19 and CD21. In blood from allergic patients, anadditional population of basophilic granulocytes are labeled by theallergen. The presence of such basophilic granulocytes is indicative ofa hypersensitive, IgE response. The enriched cell populations arefurther useful in the isolation and characterization of allergen-bindingcells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show flow cytometry plots of Paro 1-binding cells fromperipheral blood leukocytes (PBL) of a normal donor (FIG. 1B) and anallergic donor (FIG. 1A) using the MiniMACs. The specific cells weretagged with digoxigenin-conjugated Paro1 and labeled withanti-digoxigenin-microbeads and anti-digoxigenin-phycoerythrin (PE).

FIGS. 2A and 2B show flow cytometry plots of PLA₂ -binding cells fromPBL of a normal donor (FIG. 2B) and an allergic donor (FIG. 2A) with theMiniMACs. The specific cells were tagged with digoxigenin-conjugatedPLA₂ and labeled with anti-digoxigenin-microbeads andanti-digoxigenin-PE.

FIGS. 3A and 3B show counterstaining of the MACs-enriched PLA₂ -bindingcells from an allergic donor (FIG. 3A) and a normal donor (FIG. 3B) withantibodies directed against a B-cell marker (CD19) or against a markerfor basophilic granulocytes and plasma cells (CD38).

FIGS. 4A, 4B, and 4C show the phenotypic analysis of PLA₂ -binding cellsfrom an allergic donor. Positive cells are labeled with PLA₂-digoxigenin and stained with anti-digoxigenin-PE. Counterstainings areperformed with anti-CD19-fluorescein isothiocyanate (FITC) andCychrome-labeled anti-isotype antibodies. The first gate was setexcluding dead cells and monocytes (FIG. 4A). The second gate was set onthe PLA₂ -binding, PE-stained cells (FIG. 4B). Gated cells were analyzedfor surface immunoglobulin (FIG. 4C).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for the diagnosis of allergenhypersensitivity in a patient. The diagnosis is performed using apatient test sample and does not require invasive intradermal challenge.Panels of allergens may be used. Differences in the composition andnumbers of allergen binding cells in allergic vs. normal individualsprovides the basis for diagnosis. Indirect or direct conjugates of atest allergen and selection reagent, e.g. magnetic particle,fluorochrome, etc. are used to select allergen-binding cells from thesample. In normal blood, the allergen-binding cells are primarilyB-cells expressing CD19 and CD21. In blood from allergic patients anadditional population of effector cells, including basophilicgranulocytes, are labeled by the allergen. The enriched cell populationsare further useful in the isolation and characterization ofallergen-binding cells. The presence of specific allergen-bindingbasophilic granulocytes indicates hypersensitivity.

The subject methods are useful in the initial diagnosis ofhypersensitivity, and can be further used in the staging of allergicdisease and monitoring of therapy. In particular, evaluation of risk foranaphylactic shock, a life-threatening systemic reaction to allergenexposure, may be monitored. Because the subject methods analyze bloodcells, the risk of systemic reaction can be directly correlated to apositive result. Evaluation of hyposensitization treatment may assesseffector cells, e.g. basophils, eosinophils, etc., or memory B/plasmacells. Effector cell analysis may include quantitation and analysis offunctional capacity. Memory B cell analysis evaluates allergen specificpopulations according to their surface Ig class, where a shift to IgG2and other non-IL-4 induced Ig classes is indicative of successfultreatment.

The test allergen is any antigen suspected of causing a hypersensitiveimmune response. As used herein, hypersensitive immune responses arethose reactions of a mammalian immune system characterized by theproduction of high levels of IgE antibody. Contact with the allergenresults in mast cell degranulation and release of histamines, heparin,eosinophil and neutrophil chemotactic factors, leukotrienes andthromboxanes, etc. Conventional tests for hypersensitivity include askin test, where the allergen is injected intradermally. Ahypersensitive response will cause rapid production of a wheal anderythema within 30 minutes.

Allergens of interest include antigens found in food, such asstrawberries, peanuts, milk proteins, egg whites, etc. Other allergensof interest include various airborne antigens, such as grass pollens,animal danders, house mite feces, etc. Molecularly cloned allergensinclude Dermatophagoides pteryonyssinus (Der P1); Lol pI-V from ryegrass pollen; a number of insect venoms, including venom from jumper antMyrmecia pilosula; Apis millifera bee venum phospholipase A2 (PLA₂) andantigen 5S; phospholipases from the yellow jacket Vespula maculifronsand the white faced hornet Dolichovespula maculata; a large number ofpollen proteins, including birch pollen, ragweed pollen, Paro1 (themajor allergen of Parietaria officinalis) and the cross-reactiveallergen Parjl (from Parietaria judaica), and other atmospheric pollensincluding Olea europaea, Artemisia sp., gramineae, etc. Other allergensof interest are those responsible for allergic dermatitis caused byblood sucking arthropods, e.g. Diptera, including mosquitos (Anophelessp., Aedes sp., Cuffseta sp., Culex sp.); flies (Phlebotomus sp.,Culicoides sp.) particularly black flies, deer flies and biting midges;ticks (Dermacenter sp., Ornithodoros sp., Otobius sp.); fleas, e.g. theorder Siphonaptera, including the genera Xenopsylla, Pulex andCtenocephalides. The specific allergen may be a polysaccharide, fattyacid moiety, protein, etc. In many cases the allergenic epitope has beenshown to be a polypeptide. Pure allergen compositions may be isolatedfrom natural sources, prepared by expression from recombinant DNA, or beobtained by other techniques well-known in the art.

The patient may be tested with one or a panel of suspected allergens.The determination of the specific allergen to which a patient ishypersensitive allows the affected individual to seek treatment, e.g.desensitization, and to avoid activities that increase risk, e.g.exposure to the allergen. Panels may include a number of differentpollens, groups of suspected food allergens, animal allergens, etc.Samples may be run side by side, or in pools.

The subject methods are also used for the separation, culture and use ofspecific allergen-binding cells, e.g. B-cells, plasma cells andbasophilic granulocytes. The granulocytes bind the allergen through IgEantibodies that are bound to Fc receptors on the cell surface. Theenriched populations of allergen-binding B-cells are useful forproducing allergen specific antibodies, particularly IgE antibodies. Theenriched B-cells are immortalized by infection with Epstein-Barr virusor fusion with myeloma cell lines. Such B-cell lines produceallergen-specific human monoclonal antibodies, which can be used asreference reagents and for studies of repertoire and B-cell biology. TheB-cells are valuable tools to unravel the role of specific B-lymphocytesin antigen presentation.

The test allergen may be directly or indirectly conjugated to aselection reagent. In one embodiment of the invention, the selectionreagent is a magnetic reagent, such as a superparamagnetic microparticle(microparticle). Herein incorporated by reference, Molday (U.S. Pat. No.4,452,773) describes the preparation of magnetic iron-dextranmicroparticles and provides a summary describing the various means ofpreparing particles suitable for attachment to biological materials. Adescription of polymeric coatings for magnetic particles used in highgradient magnetic separation (HGMS) methods are found in DE 3720844(Miltenyi) and U.S. Pat. No. 5,385,707. Methods to preparesuperparamagnetic particles are described in U.S. Pat. No. 4,770,183.

Direct conjugation of an allergen to a magnetic particle is achieved byuse of various chemical linking groups. The polysaccharide or othercoating of the microparticle is suitably derivatized to providefunctional groups. A variety of such modifications is known in the art.Amino groups may be introduced before or after forming the beads. Analdehyde function may be introduced by reacting the polysaccharide withdiimidazol or DCCD, and coupling hexane diamine to the sugar molecules.Alternatively, in preparing the dextran for coating, aminodextran may bemixed with unsubstituted dextran to provide amino groups. Thepolysaccharides may be conveniently oxidized using periodate to providealdehyde functional groups that can be conjugated to amino substituentson a proteinaceous binding moiety, particularly under the conditions ofreductive amination. Allergens can be coupled to the particles throughside chain amino or sulfhydryl groups and heterofunctional cross-linkingreagents.

A large number of heterofunctional compounds are available for linkingto entities, Illustrative entities include: azidobenzoyl hydrazide, N-4-(p-azidosalicylamino)butyl!-3'- 2'-pyridyldithio!propionamide),bis-sulfosuccinimidyl suberate, dimethyladipimidate,disuccinimidyltartrate, N-K-maleimidobutyryloxysuccinimide ester,N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl4-azidophenyl!-1,3'-dithiopropionate, N-succinimidyl4-iodoacetyl!aminobenzoate, glutaraldehyde, and succinimidyl 4-N-maleimidomethyl!cyclohexane-1-carboxylate. A preferred linking groupis 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP)or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acidN-hydroxysuccinimide ester (SMCC) with a reactive sulffiydryl group onthe antibody and a reactive amino group on the magnetic particle.

Alternatively, in a preferred method, the allergen is indirectly coupledto the magnetic particles. The allergen is directly conjugated to ahapten, and hapten-specific, second stage antibodies are conjugated tothe particles. In this way, one magnetically coupled antibodypreparation may be used with a variety of allergens. Suitable haptensinclude digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl, avidin,biotin, etc. Methods for conjugation of the hapten to a protein, i.e.allergen, are known in the art, and kits for such conjugations arecommercially available. Empirical binding assays may be performed todetermine the optimal ratio of hapten to allergen for the subjectanalysis.

The anti-hapten antibodies may be polyclonal or monoclonal antibodies ofvarious isotypes, e.g. IgM, IgA, IgG, usually of the IgG class. Antiserais commercially available from a variety of sources, or may be raised inany convenient animal, e.g. mouse, rat, sheep, goat, etc. The antibodiesmay be coupled as intact tetramers, or fragments thereof which maintainthe specific binding portion of the molecule, e.g. Fab and F(ab')₂fragments.

In an alternative embodiment, the selective reagent is a fluorochromeconjugated allergen. Suitable labels include fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM), N,N,N',N'-tetramethyl -6-carboxyrhodamine (TAMRA), etc. Asdescribed above, the allergen may be directly or indirectly labeled.Fluorochrome reagents are useful in panel reactivity assays, where apool of two or more defined allergens, each conjugated to a differentfluorochrome, is added to a sample. A number of pools may be typed atone time, permitting a range of allergens to be tested from a singleblood draw.

A hematopoietic cell sample is taken from a patient suspected of havinga hypersensitivity to the test allergen. Conveniently, blood samples areused. The term blood sample, as used herein, shall include hematopoieticbiological samples such as blood, lymph, leukophoresis product, bonemarrow and the like; also included in the term are derivatives andfractions of such fluids. The blood sample is drawn from any site,conveniently by venipuncture. Blood samples will usually be from about 1to 100 ml of whole blood, i.e. from 10⁵ to 10⁷ nucleated blood cells,and may be treated with anticoagulants, e.g. heparin, EDTA, citrate,acid citrate dextrose or citrate phosphate dextrose, as known in theart. The sample may be subjected to treatment such as dilution inbuffered medium, concentration, filtration, or other gross treatmentthat will not involve the destruction of allergen-binding cells.

The sample may be derived from any mammal, including primate,particularly human, murine, particularly mouse, equine, bovine, ovine,porcine, lagomorpha, canine, feline, etc.

A preparation of nucleated cells may be made from the sample using anyacceptable procedure that can separate viable nucleated cells fromerythrocytes. The use of whole blood allows detection of effector cellssuch as eosinophils, in addition to basophil detection. The use ofFicoll-Paque density gradients or elutriation is well documented in theliterature. Alternatively, the blood cells may be resuspended in asolution which selectively lyses adult erythrocytes, e.g. ammoniumchloride-potassium; ammonium oxalate, etc. Treatments may also includeremoval of cells by various techniques, including centrifugation, usingFicoll-Hypaque, panning, affinity separation, using antibodies specificfor one or more markers present as surface membrane proteins on thesurface of cells, or other techniques that provide for enrichment ofleukocytes.

A directly coupled or haptenated allergen, as described above, is addedto a cell sample. The amount of allergen necessary to bind a particularcell subset is empirically determined by performing a test separationand analysis. The amount will vary with the affinity of the allergen andthe density of specific binding partner, e.g. membrane bound Ig or Igbound to a surface Fc receptor. The cells and allergen are incubated fora period of time sufficient for complexes to form, usually at leastabout five minutes, more usually at least about 10 minutes, and usuallynot more than one hour, usually not more than about 30 minutes. Ifdirectly coupled allergen is used, the incubated mixture may be directlyapplied to a magnetic separation device. If haptenated allergen is usedin the first binding step, then a magnetically coupled anti-haptenantibody is added in a second step, prior to applying the incubatedmixture to the magnetic separation device.

While not necessary for the practice of the subject methods, it may beuseful to label the cells with a fluorochrome, e.g. phycoerythrin, FITC,rhodamine, Texas red, allophycocyanin, etc. The fluorochrome label maybe used to monitor microscopically or by flow cytometry the cellcomposition after the enrichment step. Fluorescent labeling mayconveniently utilize the same indirect coupling system as the magneticparticles. For example, a hapten-coupled allergen, (e.g.digoxigenin-coupled allergen) may be used in combination with ananti-hapten antibody, (e.g. anti-digoxigenin antibody) coupled tomagnetic particles, followed by labeling with a fluorochrome conjugatedantibody directed to the anti-hapten antibody.

The medium in which the cells are separated will be one that maintainsthe viability of the cells. A preferred medium is phosphate bufferedsaline containing from 0.1 to 0.5% fetal calf serum. Various media arecommercially available and may be used according to the nature of thecells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's BasicSalt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI,Iscove's medium, phosphate buffered saline (PBS) with 5 mM EDTA, etc.,frequently supplemented with fetal calf serum, bovine serum albumin(BSA), human serum albumin (HSA), etc.

Exemplary magnetic separation devices are described in WO/90/07380,PCT/US96/00953 and EP 438,520, herein incorporated by reference. In apreferred embodiment, an improvement is provided by the use of a highgradient magnetic matrix of closely packed ferromagnetic spheres inplace of the prior art matrix of steel wool, wires, etc. The sphereswill be usually at least about 200 Tm in diameter and not more thanabout 1000 Tm in diameter, more usually at least about 250 Tm indiameter and not more than about 300 Tm in diameter. For optimumperformance it is preferred that the composition of spheres be generallyhomogeneous in size, usually varying not more than about 15% from theaverage size.

The spheres are composed of a ferromagnetic material (e.g. iron, steel,etc.), which may be coated with an impermeable coating to prevent thecontact of cells with metal. By impermeable coating it is meant apolymeric coating which contains substantially less than 30% water byweight, which does not permit the passage of ions, and which is formedon the sphere as a result of passive application, cross-linking orpolymerization of a relatively hydrophobic polymer or co-polymer.Suitable polymers include polystyrenes, polyacrylamides,polyetherurethanes, polysulfones, fluorinated or chlorinated polymerssuch as polyvinyl chloride, polyethylenes and polypropylenes,polycarbonates and polyesters, etc.

The matrix of spheres should have adequate surface area to createsufficient magnetic field gradients in the separation device to permitefficient retention of magnetically labeled cells. The volume necessaryfor a given separation may be empirically determined, and will vary withthe cell size, antigen density on the cell surface, antibody affinity,etc. The flow rate will be determined by the size of the column, butwill generally not require a cannula or valve to regulate the flow.

The labeled cells are retained in the magnetic separation device in thepresence of a magnetic field, usually at least about 100 mT, moreusually at least about 500 mT, usually not more than about 2T, moreusually not more than about 1T. The source of the magnetic field may bea permanent or electromagnet. After the initial binding, the device maybe washed with any suitable physiological buffer to remove unboundcells.

The bound cells are released from the magnetic separation means byremoving the magnetic field, and eluting in a suitable buffer. The cellsmay be collected in any appropriate medium, preferably one thatmaintains the viability of the cells. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, PBS-EDTA, PBS, Iscove's medium, etc.,which may be supplemented with-fetal calf serum, BSA, HSA, etc. Thecells may then be used as appropriate for diagnosis or antibodyproduction.

Where the sample is bound to a fluorochrome selective reagent, flowcytometry or microscopy may be used to detect the presence of B cellsand/or effector cells labeled with the allergen. Such methods arepracticed as known in the art.

The subject methods provide for an enriched population ofallergen-specific cells, including B-cells, basophilic granulocytes,eosinophils and plasma cells. In a non-allergic patient test sample, thenumber of allergen-binding cells in the enriched population may be low,due to the small absolute number of cells present in the startingpopulation. In an allergic patient test sample, the number ofallergen-specific cells present in the enriched population will usuallybe about 20%, and in some cases may be as high as 90%. The purity may beevaluated by various methods. Conveniently, flow cytometry may be usedin conjunction with light-detectable reagents specific for cell surfacemarkers expressed by leukocytes.

For a diagnosis of hypersensitivity, the enriched cell population isanalyzed for the presence of effector cells, e.g. allergen-bindingbasophilic granulocytes. Such analysis may be performed on slides, witha Coulter counter, or by flow cytometry. The expression of cell surfacemarkers such as CD38, CD25, CD7 (described in Kepley et al. (1995) J.Immunol. 154:6548-6555) or CD9 is characteristic of basophilicgranulocytes. The cells may also be characterized by staining withMay-Gruenwald/Geimsa reagents, as known in the art. Histamine release isalso used to detect basophils. Quantitative methods for histaminerelease detection include microdialysis (Peterson et al. (1992) J.Allergy 47:635-637); HPLC (Leino et al. (1990) Agents Actions31:178-182); immunoassay (Hammar et al. (1990) J. Immunol. Methods128:51-58); microfibre method (Nolte et al. (1987) Allergy 42:366-373),etc.

In an allergic patient, at least about 50% of the allergen-binding cellswill be effector cells such as basophilic granulocytes, and may be ashigh as 90% of the allergen-binding cells. In a non-allergic patient,less than about 10% of the allergen-binding cells are basophilicgranulocytes. A positive diagnosis of allergy to a specific antigen ismade when the basophil population is increased relative to a controlsample. The number of basophils may be at least about twice that of anormal, non-allergic donor in a similarly treated sample, and may be ashigh as about ten times the number of basophils in a control sample.

Allergen-binding B-cells from the enriched cell population, particularlyB cells from human donors, may be used to produce allergen-specificantibodies. The B cells may be immortalized through infection withEpstein-Barr virus, fusion with a myeloma cell line, transfection with atransforming retrovirus, etc. to produce a monoclonal cell line.Screening for cell lines that produce allergen-specific antibodies isperformed by any convenient method, e.g. ELISA, RIA, etc. to determineallergen specificity. B-cells that produce monoclonal IgE are ofparticular interest for the production of testing reagents, etc.

A kit may be provided for the practice of the subject invention. Forexample, the kit may include one or a panel of hapten-conjugatedallergens (for example a series of digoxigenin-coupled pollen, insectand/or food allergens), and either magnetic particles conjugated toanti-hapten antibodies or fluorescent labeled anti-hapten antibodies.The kit may further comprise a means for magnetic separation, andbuffers and other reagents used in the separation process.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Materials and Methods

Antigens and preparation of conjugates. Phospholipase A2 from bee venomwas purchased from Sigma, Freiburg, Germany (P 9279). Paro1 was purifiedas described in Oreste et al. (1991) Int. Arch. Allergy Immunol.96:19-27. Both allergens were conjugated to digoxigenin (dig), using theDIG antibody labeling kit from Boehringer Mannheim, FRG. Preparationswere made at different protein to digoxigenin molar ratios, and testedby titration on peripheral blood mononuclear cells (PBMC) for theirability to stain specifically a distinct population of the PBMC, but notmonocytes or macrophages.

Cells. Blood was obtained from normal donors (blood bank of theUniversity of Cologne) or from allergic patients with a known history ofsevere allergy either against bee venom or Parietaria officinalis.Nearly all of the allergic patients had elevated levels ofallergen-specific IgE serum antibodies. Patients allergic against beevenom all showed a positive reaction in skin tests performed to confirmthe diagnosis.

Magnetic and fluorescent labeling of cells with antigen. Blood sampleswere diluted with two volumes of RPMI, and aliquots of 30 ml werelayered onto 15 ml of Ficoll-Hypaque (Pharmacia, Freiburg, FRG). Aftercentrifugation (for 20 minutes at room temperature, 800 g) cells at theinterphase were collected and washed three times with cold phosphatebuffered saline (PBS). Cells were resuspended in a small volume ofPBS/0.5% BSA/10 mM EDTA, and incubated for 10 minutes on ice with 5mg/ml of the murine monoclonal antibody A20/44 (IgG₁) to blocknonspecific binding. Allergen-digoxigenin conjugates were added at 10Tg/ml and the cells were incubated for another 10 minutes. After washingwith PBS/BSA/EDTA, the cells were then incubated (for 10 minutes at roomtemperature) with superparamagnetic microbeads coupled toanti-digoxigenin antibodies (Miltenyi Biotec, Bergisch-Gladbach,Germany), making a 1/5 dilution of microbeads to cell suspension.Finally, the cells were stained with 1/50 volume ofStreptavidin-PE-coupled anti-digoxigenin antibodies by incubation for 10minutes on ice, and finally washed again. Prior to magnetic separation,the cells were deaggregated by passage through a 40 Tm nylon-mesh(Partec, Munster, FRG0).

Antigen-specific magnetic cell sorting. Magnetic isolation was performedessentially as described in Miltenyi et al. (1990) Cytometry 11:231-236.Magnetic separation MiniMACs columns (Miltenyi Biotec, GmbH) wereprewashed with 4 ml of degassed PBS/BSA. Half of the cell sample wasloaded onto the column, the column was washed, and the other half of thesample was applied. Then, the columns were washed with 5 ml PBS/BSA.Cells which had bound to the allergen-digoxigenin conjugate, and therebybound the antibody-coupled magnetic microbeads, were retained in theseparation column in the presence of a magnetic field. To elute theretained cells, the columns were removed from the magnetic field and thecells were washed from the column with 1 ml of PBS/BSA. The separationprocedure was monitored by flow cytometry using a FACScan and FACScanResearch or Cellquest software (Becton Dickinson, San Jose, Calif.). Forcytometric analysis, 10 Tg/ml of propidium-iodide (PI) was added inorder to identify dead cells not only according to forward versus sidescatter, but also according to relative F2 versus F3 fluorescence.

Immunofluorescence. The following antibodies were used forimmunophenotyping: ICD3, ICD5, ICD10, ICD14, ICD16, ICD19 (described inAllison and Lanier (1987) Ann. Rev. Immunol. 5:503-540; Gadol and Ault(1986) Immunol. Rev. 93:24-34; Pezzutto et al. (1987) J. Immunol.138:2793-2799; Perussia et al. (1984) J. Immunol. 133:180-189; Bhan etal. (1981) J. Exp. Med. 154:180) conjugated to fluoresceinisothiocyanate (FITC), or phycoerythrin (PE), all obtained from BecktonDickinson. ICD9, ICD21, ICD23, ICD38, (described in Nadler et al. (1983)J. Immunol. 131:244-250; Tedder et al. (1984) J. Immunol. 133:678-683;Thorley-Lawson et al. (1985) J. Immunol. 134:3007-3012; Uchiyama et al.(1981) J. Immunol. 126:1398-1403) were all obtained from Immunotec(Dardilly, France) as FITC-conjugates. The isotype-specific antibodieswere all purchased as biotin (Bio)-, or horseradish-peroxidase (HRPO)conjugates. IIgG1-Bio, IIgG4-Bio, IIgE-Bio, IIgG-HRPO, IIgM-HRPO,IlgE-HRPO, IKappa-HRPO, ILambda-HRPO were obtained from SBA,(Birmingham, Ala.). IDigoxigenin-beads and IDigoxigenin-PE were obtainedfrom Miltenyi Biotec (Bergisch Gladbach, FRG). Streptavidin-Cychrome waspurchased from Pharmingen (San Diego).

Generation of EBV-lines. Feeder cells were generated from autologousPBMC by depletion of B-lymphocytes with ICD19-superparamagneticmicrobeads and high gradient magnetic cell sorting using an A2 column(Miltenyi Biotec, Bergisch Gladbach, FRG) with a 0.4 Tm needle. Thenegative cells were incubated for 30 minutes at 37° C. with 50 Tg/mlmitomycin C (Medac, Hamburg, FRG). To remove excess mitomycin beforeadding the feeder cells to the sorted cells, the prospective feedercells were washed three times in 50 ml of RPMI, supplemented with 5%FCS. After the last washing step, T-lymphocytes were suppressed byadding 1 Tg/ml cyclosporin A (Sandoz, Basel, Switzerland), and culturedin the medium described below. EBV-lines were generated from the antigenpositive fractions of the magnetic sorts by incubating 100-1000 cellsper well in 96-well flat bottom plates (Costar, Mass., USA) with 1/4volume of supernatant of the EBV secreting marmoset cell line B95-8(Miller and Lipman (1973) P.N.A.S. 70:190-194). These cells were layeredonto 2×10⁵ autologous feeder cells per well, generated from PBMC asdescribed above, cultured in RPMI with 10% FCS, 50 U/ml penicillin, 50Tg/ml streptomycin, and 2 mM L-glutamine. After two weeks, aggregatesrepresenting the progeny of single cells were picked under themicroscope and diluted over a series of wells. For most of the linesgenerated this way, monoclonality was later confirmed by restrictionanalysis of JH-rearrangements.

Western Slot Blots. Different amounts of unconjugated PLA₂, or bovineserum albumin (BSA) as negative control, were blotted ontonitrocellulose-filters (Hybond C extra, Amersham, Braunschweig, FRG),using a micro-sample-filtration manifold (Schleicher-Schuell, Dassel,FRG). After washing with PBS, the filters were blocked for one hour withTrizma-Base, NaCl, pH 7.6 containing 3% Tween 20 (TBS) (Serra,Heidelberg FRG), and 5% milk powder. The culture supernatants of theEBV-lines generated were deaggregated and sterilized by filtration,diluted with one volume of TBS/Tween/milk powder and applied to thefilters. After incubation for one hour, filters were washed three timesfor five minutes in TBS/Tween20. Then the filters were incubated withHRPO-conjugates of antibodies, specific for the various human isotypesand diluted in TBS/Tween/drymilk, for 40 minutes at room temperature.After washing, as described before, filter-bound HRPO was quantitatedusing the ECL-System (Amersham, Braunschweig, FRG). Autoradiographs wereobtained on X-OMAT-AR-films (Eastman-Kodak, New York, USA).

DARIA (double antibody radioimmunoassay). The DARIA was essentiallycarried out as described in Platts-Mills et al. (1978) J. Immunol.120:1201-1210. For the determination of Paro1 specific human IgE or IgG;50 Tl of supernatant from EBV-lines were mixed with 50 Tl of anappropriate dilution of an IgE or IgG myeloma protein. This solution wasincubated for four hours at room temperature with 100 Tl of boratebuffered saline (BBS, pH 8.0) containing 1 ng of radiolabeled Paro1(10-20,000 cpm). Antigen-antibody complexes were precipitated byaddition of an appropriate amount of goat anti-human IgE or IgG for 16hours at 4° C. Precipitates were collected on Whatman GF/A filters.After washing the filters in 10 ml of BBS, the bound radioactivity wasdetermined in a gamma counter.

Amounts of antibodies were expressed as: ((a-b)/(B_(max) -b)%), wherea=cpm bound by the tested supernatant, b=background cpm (cpm bound by apool of sera without IgE against Paro1) and B_(max) =cpm bound by thepositive reference serum pool (a pool of sera with high levels of Paro1specific IgE). Supernatants binding less than 1.3×background cpm wereconsidered negative.

May-Gruenwald-/Giemsa-staining After MACS-separation, the fractionsenriched for allergen-binding cells were resuspended in 300 Tl of RPMI1640 supplemented with 30% FCS and collected by centrifugation for 10minutes at 400 g at 4° C. The pellets were resuspended in 100 TlRPMI/30%/FCS. Cytospins were prepared in a Cytospin centrifuge(Shahdon-Elliott), spinning the cells onto the slides at 1000 rpm forthree minutes. The cytospin preparations were air-dried overnight atroom temperature. The slides were then incubated for five minutes inMay-Gruenwald solution (Merck, Darmstadt, FRG), followed by incubationin a 1:1 dilution of May-Gruenwald:Weisebuffer for another five minutes.The final staining step was carried out by immersing the slides in a1:10 dilution of Giemsa solution (Merck Darmstadt, FRG) in water for 15minutes. The slides were then washed in distilled water and air driedfor 30 minutes. Finally, the preparations were embedded in Eukitt(Kindler Freiburg, FRG), and covered with a cover glass, and analyzed ina Zeiss Axiolab microscope.

Results

Enrichment of allergen-binding cells. For the isolation ofallergen-binding cells from peripheral blood (50 ml from allergic donoror 500 ml from normal donors), the blood was depleted of erythrocytes,neutrophils and eosinophils on a Ficoll-gradient. The mononuclear cellswere then stained with digoxigenin-conjugates of either PLA₂ or Paro1,anti-dig-MACS microbeads and anti-dig-antibodies conjugated tophycoerythrin. As determined by flow-cytometry, the frequency ofallergen-binding cells was below 0.1% in the blood of normal donors,whereas samples from allergic donors contained between 0.4% and 2.3% ofallergen-binding cells before MACS-separation. The percentages ofallergen-binding cells were calculated by analysis of 100,000 events byFACS. The allergen-binding cells were enriched by high gradient magneticcell sorting with the MiniMACS to frequencies of up to 75% (allergicdonors) and 2-45% (non-allergic donors). The data are shown in FIGS. 1and 2. From some allergic donors (50 ml of blood), up to 8×10⁵allergen-binding cells, mostly basophilic granulocytes were enriched,with only 3-11% B-cells. From the blood of normal donors (500 ml), atmost 6×10⁵ allergen-binding cells were enriched, nearly all of themB-lymphocytes.

Phenotype of the allergen-binding cells. The indirect staining of thedigoxigenin-conjugated allergen with anti-dig-PE was used to monitor themagnetic cell sortings. It also allowed the phenotypical analysis of thesorted allergen-binding cells by multiparameter flow cytometry. Thecells were counterstained with fluorescein- and CyChrome-conjugatedantibodies specific for various surface markers, with the remainingPE-negative cells serving as an internal control. The allergen-bindingcells from both normal and allergic donors did not express CD3, CD14 orCD16, markers for T-cells, monocytes or NK-cells. The frequency oflabeled cells was very low. Cell types known for their ability to bindfluorochrome conjugates non-specifically were not enriched by theprocedure.

A drastic difference was observed when comparing allergen-binding cellsfrom the blood of allergic and non-allergic donors with respect to theexpression of CD19, a B-cell marker, and CD38, a marker of plasma cellsand basophilic granulocytes. The absolute numbers of allergen-bindingCD19⁺ B-cells were about the same. However, about 90% of theallergen-binding cells enriched from the blood of normal donors, butonly 3-12% from allergic donors are CD19⁺ B-cells. The data are shown inFIG. 3. Almost all of these cells also express CD21, the receptor forEBV, making them a suitable target for transformation and to generateallergen-specific B-cell lines. These results are summarized in Table 1.

                                      TABLE 1    __________________________________________________________________________    normal donor            allergic donor    1        2    3    4    Z1   ET   BO   GA    __________________________________________________________________________    before         0.02%              0.1%                   0.03%                        0.08%                             1.89%                                  1.75%                                       0.68%                                            0.36%    MACS    after        12.2%             18.2%                   4.7%                       45.8%                            92.2%                                 44.8%                                      60.0%                                           23.8%    MACS    CD38         3.0%              9.1%                  12.1%                        9.6%                            93.2%                                 72.0%                                      90.9%                                           82.0%    CD19        95.1%             82.0%                  78.5%                       97.7%                             2.8%                                 11.8%                                       7.5%                                           11.3%    Ratio        90.0%             81.2%                  71.5%                       95.3%                             2.6%                                 12.1%                                       6.2%                                           10.7%    CD19/    CD21    __________________________________________________________________________     The numbers show the percentages of PLA.sub.2binding cells from blood of     normal and allergic donors before and after enrichment with MACS. For     phenotypical analysis the PLA.sub.2specific cells were counterstained wit     antiCD38-FITC or antiCD19-FITC. The results of a three color analysis,     staining the cells with PLA.sub.2PE, antiCD19-Cychrome and antiCD21-FITC     are also shown.

The majority of the enriched allergen-binding cells from blood ofallergic donors stain positively for CD38, a marker expressed primarilyon plasma cells and basophilic granulocytes. These cytometric analysestogether with the May-Gruenwald/Giemas staining, clearly identify thesecells as basophilic granulocytes. They represent approximately 72-95% ofthe enriched, allergen-binding cells, whereas in the case of normalblood donors only a few of these cells are observed. The specificantigen-receptor of basophilic granulocytes is most likely aFc-receptor-bound immunoglobulin. Accordingly, nearly all of theallergen-binding CD38⁺ cells stain for surface IgE, some also for IgG4,but none for IgG1 (shown in FIG. 4). Some of the enrichedallergen-binding cells were transformed with Epstein-Barr virus. ThreeEBV-lines produce IgG, and two lines produce IgM specific for PLA₂, aswas determined by Western Blot. Several lines produce Paro1-specificantibodies, as determined in Paro1-specific double antibody radioimmunoassay (DARIA). Out of 25 EBV-lines tested, six producedParo1-specific IgE, and several others produced differentIgG-subclasses.

It is evident from the above results that the subject invention providesa rapid method for isolating allergen-binding cells from normal orallergic patient test samples. In the blood of allergic individuals, ahigh number of CD38-expressing allergen-binding cells are found. ByMay-Gruenwald and Giemsa staining, they are identified as basophilicgranulocytes, which also stain with CD9, and weakly with CD25. The clearstaining of basophilic granulocytes offers new means of allergydiagnosis and monitoring. The cellular analysis of the reactive effectorcells has clear advantages over the commonly used provocation tests,since it does not have the side effects and risks for the patients.Furthermore, this technique provides more detailed information about thecell subsets and relative numbers of allergen-binding cells.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto, such as the use of any suitableseparation means, without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A method of producing allergen-specificmonoclonal antibody, the method comprising:a. combining a hematopoieticcell sample with a test allergen directly or indirectly coupled to amagnetic microparticle; b. passing said cells through a magnetic field;c. removing unbound cells; and d. collecting bound cells in thesubstantial absence of said magnetic field to provide an enriched cellsample comprising allergen binding cells; e. immortalizing B-cells insaid enriched cell sample; and f. screening said immortalized B-cellsfor cells that produce allergen-specific antibody.
 2. The method ofclaim 1, wherein said allergen-specific antibody is an IgE antibody. 3.The method of claim 1, wherein said hematopoietic cell sample is a bloodsample.
 4. The method of claim 1, wherein said allergen is a protein. 5.The method of claim 4, wherein said protein is conjugated to a hapten,and anti-hapten antibody conjugated to a magnetic microparticle isincluded in said combining step.
 6. The method of claim 1, wherein saidimmortalizing step comprises infection with Epstein-Barr virus.